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Molecular Diagnostic Solutions in Algal Cultivation Systems
Published in Stephen P. Slocombe, John R. Benemann, Microalgal Production, 2017
Laura T. Carney, Robert C. McBride, Val H. Smith, Todd W. Lane
In cases where a deleterious organism or weed species is of sufficient abundance in the contaminated culture or has been isolated or enriched in culture, it is possible to clone and carry out dideoxynucleotide terminator sequencing (Sanger et al. 1977) of the desired region. With Sanger sequencing, it is generally possible to achieve 700 nt of sequence data per primer extension reaction. Thus, paired-end reactions should be sufficient to sequence both DNA strands of the ITS region and single strands of most, if not all, of the full-length SSU rRNA genes. By providing full sequencing coverage of the region of interest, Sanger sequencing can result in a high degree of taxonomic distinction of the target organism and maximal information for the design of PCR primer or oligonucleotide probes for the future detection and quantification of the deleterious species in algal mass culture.
Biomems
Published in Simona Badilescu, Muthukumaran Packirisamy, BioMEMS, 2016
Simona Badilescu, Muthukumaran Packirisamy
As shown in Figure 9.1, biomolecule arrays are divided into three categories: antibody arrays, protein/peptide arrays, and DNA arrays. DNA arrays are used to characterize cDNA libraries by DNA hybridization with single DNA probes, and to determine gene expression patterns by hybridization with complex hybridization probes. Multiplexed genotyping is achieved by primer extension of arrayed oligos using reverse transcription with complex RNA mixtures as the transcription templates. The main applications of arrays of antibodies, RNA aptamers, or plastibodies with known binding specificities will be used for the detection and quantification of biomolecules, such as proteins, peptides, or chemical compounds from complex mixtures such as clinical samples. In contrast, arrays consisting of recombinant proteins or synthetic peptides are mainly used to identify and characterize interactions or biological activities of proteins with various kinds of biomolecules. An example could be the screening of an arrayed expression library with an antibody of unknown binding specificity.
Artificial Enzymes
Published in Yubing Xie, The Nanobiotechnology Handbook, 2012
James A. Stapleton, Agustina Rodriguez-Granillo, Vikas Nanda
In the traditional in vitro selection process, there is no room to select for advanced enzymatic properties such as multiple turnover, since selection is generally based on formation of a covalent linkage between the RNA and a substrate labeled with a “capture” tag (Silverman 2009). One way of getting around this limitation is to use IVC strategies (Tawfik and Griffiths 1998), in which the sequence of the ribozyme (genotype) and its catalytic activity (phenotype) become “linked” within individual droplets in a water-in-oil emulsion. Recently, a novel selection approach based on this strategy was developed to engineer an RNA polymerase capable of synthesizing RNAs of up to 95 nucleotides (Wochner et al. 2011). The method is termed compartmentalized bead-tagging, and consists of encapsulating a genetic library of ribozymes attached to magnetic beads in individual droplets, allowing transcription to occur within the droplets to create the ribozymes, triggering primer extension by addition of primer/template duplexes in a second emulsion, and detecting the extent of primer extension by a combination of rolling circle amplification of the extended primers and fluorescence-activated cell sorting (FACS; Figure 3.5). This method, in combination with rational RNA engineering, yielded an RNA polymerase with greater polymerase activity, fidelity, and generality than the parental ribozyme. The new ribozyme was able to synthesize an enzymatically active ribozyme from an RNA template (Wochner et al. 2011), and can polymerize sequences half of its own length, bringing us closer to the goal of a completely self-replicating ribozyme.
Development of amplification system for point-of-care test of nucleic acid
Published in Computer Methods in Biomechanics and Biomedical Engineering, 2022
Shaolei Huang, Jiageng Wu, Haozheng Dai, Runxin Gao, Hongyu Lin, Dongxu Zhang, Shengxiang Ge
The amplification system proposed in this paper is based on the principle of PCR, so the main technical requirements should meet the temperature conditions of the three processes, which are denaturation, annealing and extension. High-temperature denaturation process needs to be carried out at the temperature of 95 °C to open the double helix structure. Detection of RNA is slightly different from DNA. The temperature condition of 50 °C needs to be added for its reverse transcription; During low temperature annealing, the primer and the single strand are combined according to the principle of base pairing, and the temperature should be maintained at 55 °C; Primer extension process requires temperature condition of 72 °C to achieve the synthesis of complementary strands, and the nucleic acid polymerase will extend along the direction of phosphate (5′-3′) (Harve et al. 2010). It has been shown that if the target nucleic acid fragment to be amplified is short (100-300 bp), a two-step PCR can be used, namely two temperature conditions where the extension phase can occur during the transition between annealing and denaturation temperatures and does not require a holding time (Wittwer and Garling 1991, Wittwer et al. 2001). Generally, the above process needs to be repeated about 40 to 50 cycles and the temperature conditions between the cycles should be consistent. In addition, to meet the requirements of POCT, it is necessary to complete at least 45 cycles of the process within 30 minutes, that is, the average temperature change velocity is not less than 4 °C/s.